Adeno-associated virus type 2 is an apparently non-pathogenic human virus that can infect cells in a cell cycle-independent fashion. The characteristics of AAV have been exploited in extensive efforts that have shown AAV to be among the most promising therapeutic gene transfer vehicles. In addition to the productive life cycle of AAV (that requires the present of a helper virus such as adenovirus or herpes viruses) AAV has evolved the unique ability to integrate its genome site- specifically into human chromosome 19 in order to establish a latent infection. Our long-term goal is to elucidate the molecular mechanisms underlying AAV2 replication, site-specific integration and packaging. The small genome of AAV contains two open reading frames (REP and CAP). The Rep proteins orchestrate all aspects of the viral life style including DMA replication, site-specific integration, rescue from the integrated state and virus packaging. The study of this protein has been limited to biochemical characterizations and the lack of structural information has prevented a comprehensive depiction of the mechanistic aspects of Rep-mediated processes. Recently, we have solved the structure of the core helicase domain of AAV2 Rep. As this motor domain is present in all four Rep proteins it is likely to play an essential role in all aspects of Rep function. In this application we propose a collaborative and multifaceted approach (combining structural, biophysical and biochemical studies) aimed at understanding the structural basis for the motor activity of Rep within the context of the different isomers. Specifically, we will investigate the structural determinants of the Rep40 ATP-dependent motor activity (Aim 1); we will complement and extend the structural information by biochemical studies designed to help develop a model for the mechanism of Rep's motor activity (Aim 2); and, using Rep68, we will study the utilization of the motor activity that, in this context, is directed at replicative DMA unwinding. (Aim 3).